The Hepatitis C virus (HCV) causes one of the worlds most pandemic and insidious diseases. According to the World Health Organization, there are approximately 170 million carriers worldwide with a prevalence up to 0.5-10% (Lancet 351:1415 (1998)), while in the United States, almost four million individuals are afflicted (Alter and Mast, Gastroenterol. Clin. North Am. 23:437-455 (1994)). Unfortunately, 75-85% of people infected will develop a chronic infection, which may ultimately lead to cirrhosis and hepatocellular carcinoma in 10-20% and 1-5%, respectively, of chronically infected people (Cohen, Science 285:26-30 (1999)).
The causative agent, hepatitis C virus, was identified in 1989 and has accounted for 50-60% of the non-A, non-B transfusion associated hepatitis (Alter, et al., N. Engl. J. Med. 321: 1494-1500 (1989); Choo, et al., Science 244:359-362 (1989); Kuo, et al., Science 244:362-364 (1989)). More than 100 strains of the virus have since been identified, and have been grouped into six major genotypes that tend to cluster in different regions of the world (Simmonds, Current Studies in Hematology and Blood Transfusion, Reesink, ed., Karger, Basel, pp. 12-35 (1994); van Doorn, J. Med. Vir. 43:345-356 (1994)).
HCV, a member of the Flaviviridae family, is a positive-sense, single-stranded RNA virus with genome size of approximately 9.6 kb (Heinz, Arch. Virol. Suppl. 4:163-171 (1992); Mizokami and Ohba, Gastroenterol. JPN 28 Suppl5:42-44 (1993); Ohba, et al., FEBS Lett. 378:232-234 (1996); Takamizawa, et al., J. Virol. 65: 1105-1113 (1991). The genomic RNA encodes a polyprotein of approximately 3000 amino acid residues in the order of NH2—C— E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B—COOH (Lohmann, et al., J. Hepatol. 24: 11-19(1996); Simmonds, Clin. Ther. 18 Suppl. B:9-36 (1996). The polyprotein undergoes subsequent proteolysis by host and viral enzymes to yield the mature viral proteins (Grakoui, et al., J. Viral. 67:1385-1395 (1993); Shimotohno, et al., J. Hepatol. 22:87-92 (1995). HCV NS5B is essential for the virus replication and is a viral coded RNA dependent RNA polymerase carrying various functions, including copying the negative strand from the positive strand, and generating multiple copies of positive infectious RNA from the negative template strand.
To date, interferon-alpha and newer versions of longer lasting pegylated interferon monotherapy as well as in combination therapy with ribavirin (Rebetron, Schering-Plough, Kenilworth, N.J.) are among the few approved treatments of hepatitis C. However, typically less than 10% of patients respond to interferon-alpha monotherapy, and 41% of patients respond to combination therapy (Reichard, et al., Lancet 351:83-87 (1998). Therefore, it has become increasingly important to develop more effective antiviral agents against the various viral targets to combat hepatitis C. Among the most promising antiviral targets in chronic HCV infection are the replication enzymes, RNA-binding proteins, viral entry proteins and enzymes required for the virus' maturation processes, and many of those targets have been thoroughly investigated.
For example, the crystal structure of the HCV RNA dependent RNA polymerase has been determined to help design inhibitors for this enzyme. Expectedly, NS5B has been found to share various canonical features of other polymerases. Among other things, NS5B is folded from a single peptide chain into an overall shape of a right hand with three sub-domains: palm, thumb and fingers. The catalytic residues Asp318, Asp319 and Asp220 are located at the palm and encircled by palm, thumb and finger domains, while the thumb and finger domains are interconnected through extended loops. The channel between thumb and fingers define the RNA template-binding site. The NTP is fed through the back channel defined by the three domains and a linkage between thumb and fingers. The duplex RNA is released through a channel on the opposite side of the NTP channel. One particular structural feature in the HCV RNA polymerase is an anti-parallel β-loop that extends from the thumb domain toward the finger domain and is unique among all known polymerases. The cavity between this loop and the active site define the RNA primer-binding site (i.e., initiation nucleotide binding site for HCV). However, despite the relatively extensive knowledge of the molecular architecture of NS5B, design of suitable inhibitors has significantly lagged behind the expectations. For example, targeting of the initiation nucleotide binding site appeared not to be sufficiently promising to many groups for designing of an inhibitor, since the affinity of this site for its natural substrate (the initiation nucleotide) is already relatively low (in the range of 10−3˜10−4 M).
Alternatively, chemical modification of enzymes may be a useful tool in designing drugs that interrupt the catalytic activity of enzymes (e.g., suicide inhibitors). Chemical modification of enzymes to alter their specificity and catalytic activity has been studied for many years (Per Berglund, Grace DeSantis, Michele R. Stabile, Xiao Shang, Marvin Gold, Richard R. Bott, Thomas P. Graycar, Tony Hing Lau, Colin Mitchinson, J. Brayan Jones, J. Am. Chern. Soc. 1997, 119,5265-5266; Kaiser, E. T. Acc. Chern. Res. 1989,22,47-54; Neet, K. E.; Koshland, D. E., Jr. Proc. Natl. Acad. Sci. U.S.A., 1966,56, 1606-1611; Peterson, E. B.; Hilvert, D. Biochemistry 1995, 34, 6616-6620). For example, in one approach cysteine is introduced at a specific position and then reacted with thiosulfonate reagents for the modification of proteins (Kenyon, G. L.; Bruice, T. W. Methods Enzymol. 1977,47,407-430; Wynn, R.; Richards, R. M. Methods Enzymol. 1995, 251, 351-356; Roger L. Lundblad, “Chemical Reagents for Protein Modification”, CRC Press, 1991, Chapter 6, pages 59-93). However, the sulfhydryl group of cysteine is a relatively reactive functional group in proteins and may therefore readily and often indiscriminately react with various cysteine modifying agents (e.g., cysteinyl residues are easily alkylated, aceylated and arylated).
Modification of cysteine residues has historically been utilized to improve the selectivity and catalytic activity of proteins, or to inhibit selected enzymes, and particularly cysteine proteinases (see e.g., Curr. Med. Chem. 2002, May 9 (9):979-1002 “Thiol-dependent enzymes and their inhibitors: a review”). However, while various enzymes could be modified using thiol-specific agents, there is to the best of the inventors knowledge no known example of cysteine modification that was reported to inhibit activity of viral enzymes, and especially viral polymerases.
Therefore, although numerous viral polymerase inhibitors are known in the art, all or almost al of them suffer from various disadvantages. Thus, there is still a need to provide compositions and methods to inhibit viral polymerases, and especially the HCV de-novo RNA polymerase.